image quantification

[Benito Alarcon]

Dear all,
We would like to know your opinion about the following
experiment.  We try to know the amount of a protein present in the
cytoplasm that can go to the nucleus. The experiment is done in C.
elegans that express our protein linked to RFP and a nuclear marker
linked to GFP (used to draw the roi of the nucleus). We have taken
the RFP and GFP images with automatic exposure in a fluorescence
microscope.  The images were then analyzed using Roi Manager:  we
measure the roi of the nucleus and the roi of the complete cell in
the RFP image. Then, the rawintden value of the nucleus was
represented as a percentage of the total signal (the rawintden of the
complete cell) and considered the part of the signal that goes to the
nucleus. Since we are not experts in image quantification, any
critic/opinion from you will be appreciated.
Thanks
B. Alarcon
Instituto de Biomedicina de Valencia-CSIC
Valencia, Spain.

--
ImageJ mailing list: http://imagej.nih.gov/ij/list.html

[John Oreopoulos]

Dear Benito,

I would recommend reading the following as a starting point for this kind of analysis:

Pawley, J., The 39 steps: A cautionary tale of quantitative 3-D fluorescence microscopy. Biotechniques, 2000. 28(5): p. 884-888.
North, A.J., Seeing is believing? A beginners' guide to practical pitfalls in image acquisition. Journal of Cell Biology, 2006. 172(1): p. 9-18.
Waters, J.C., Interpreting fluorescence microscopy images and measurements. Cell, 2008.
Waters, J.C., Accuracy and precision in quantitative fluorescence microscopy. Journal of Cell Biology, 2009. 185(7): p. 1135-1148.

Then, after that, take a look at the excellent tutorials on the ImageJ Wiki and the FIJI website (and others as well):

http://imagejdocu.tudor.lu/doku.php?id=video:start
http://pacific.mpi-cbg.de/wiki/index.php/Colocalization#Colocalization_Analysis_-_What_is_Colocalization_anyway.3F
http://rsbweb.nih.gov/ij/docs/examples/index.html
http://www.fmhs.auckland.ac.nz/sms/biru/_docs/Image_Analysis_Basics.pdf

Sincerely,

John Oreopoulos
Staff Scientist
Spectral Applied Research Inc.
A Division of Andor Technology
Richmond Hill, Ontario
Canada
www.spectral.ca



On 2014-06-02, at 6:35 AM, Benito Alarcon wrote:

> Dear all,
> We would like to know your opinion about the following experiment.  We try to know the amount of a protein present in the cytoplasm that can go to the nucleus. The experiment is done in C. elegans that express our protein linked to RFP and a nuclear marker linked to GFP (used to draw the roi of the nucleus). We have taken the RFP and GFP images with automatic exposure in a fluorescence microscope.  The images were then analyzed using Roi Manager:  we measure the roi of the nucleus and the roi of the complete cell in the RFP image. Then, the rawintden value of the nucleus was represented as a percentage of the total signal (the rawintden of the complete cell) and considered the part of the signal that goes to the nucleus. Since we are not experts in image quantification, any critic/opinion from you will be appreciated.
> Thanks
> B. Alarcon
> Instituto de Biomedicina de Valencia-CSIC
> Valencia, Spain.
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html


--
ImageJ mailing list: http://imagej.nih.gov/ij/list.html